Chromosomal Analysis By Molecular Karyotyping

ABSTRACT

The invention provides a method of karyotyping (for example for the detection of trisomy) a target cell to detect chromosomal imbalance therein, the method comprising: (a) interrogating closely adjacent biallelic SNPs across the chromosome of the target cell (b) comparing the result at (a) with the SNP haplotype of paternal and maternal chromosomes to assemble a notional haplotype of target cell chromosomes of paternal origin and of maternal origin (c) assessing the notional SNP haplotype of target cell chromosomes of paternal origin and of maternal origin to detect aneuploidy of the chromosome in the target cell. Also provided are related computer-implemented embodiments and systems.

TECHNICAL FIELD

The present invention relates generally to methods and materials for usein detecting abnormalities of the number of whole chromosomes orchromosome regions (aneuploidy). It has particular utility for prenataldiagnosis, either before pregnancy is established in gametes and cellstaken from early embryos or later in pregnancy in samples of cells fromthe placenta or fetus.

BACKGROUND ART

In normal meiosis the precursor cells of the sperm or ova must multiplyand then reduce the number of chromosomes to one half set in each gametein two specialised meiotic divisions. During the early stages of meiosisfollowing DNA replication, the four duplicated chromatids of ahomologous pair align closely along their length and may exchangesegments, resulting in non-recombinant (no exchange) and recombinantchromosomes and generating genetic variation. The resultant gametes,therefore, each contain a single chromosome which is either arecombinant of both homologous chromosomes or is non-recombinant andidentical to one of the parental chromosomes. This is shown in FIG. 1(a).

Aneuploidy is defined as an abnormal number of whole chromosomes orparts of chromosomes causing a genetic imbalance which may be lethal atearly stages of development, cause miscarriage in later pregnancy orresult in a viable but abnormal pregnancy. The most frequent andclinically significant aneuploidies involve single chromosomes (strictly‘aneusomy’) in which there are either three (‘trisomy’) or only one(‘monosomy’) instead of the normal pair of chromosomes.

In early development, aneuploidy can arise through abnormal chromosomalsegregation following replication and cell division either (1) duringthe two meiotic divisions which normally result in a haploid, half set,in each gamete before fertilisation, or (2) during the early divisionsof the cleavage stage fertilised embryo. FIG. 1( b) shows the effects ofnon-disjunction ie failure of replicated chromosomes to separate duringdivision, which is a common cause of abnormal chromosome segregation,during the two meiotic divisions.

The aim of molecular karyotyping is to identify numerical or structuralchromosomal abnormalities and in particular to identify any imbalanceinstead of the normal two copies of a chromosome or chromosome segment.

Currently there are two basic approaches to molecular karyotyping:

The first is to use molecular genetic markers, often highly polymorphicshort tandem repeats (STRs), for each of the parental chromosomes. Wherethere is a different repeat on each of the parental chromosomes the STRmarker is fully informative i.e. capable of identifying the presence ofeach chromosome (at that position). By use of a number of STR markers,the confidence in the method can be improved. An example of the use ofSTRs in trisomy analysis is given by Findlay et al. (1998) Journal ofAssisted Reproduction and Genetics Vol 15, No 5: 1998: 266-275.

The second approach, comparative genomic hybridisation (CGH), involvesfluorescent labelling of test and normal genomic DNA and comparison ofquantitative differences in chromosome specific sequences by hybridisingeither to metaphase chromosomes or DNA clones on a microarray (arrayCGH). Generally test and reference DNA is labelled with differentfluorochromes and hybridised to the DNA microarray with a series ofcloned target DNAs, for example, BAC clones, derived from particularchromosomal regions. Such ‘chips’ are tailored to prenatal diagnosis andother diagnostic applications by including BACs informative, forexample, for common deletion syndromes as well as aneuploidy andunbalanced translocations.

Given the importance of karyotyping, it can be seen that novel methodsand materials relating to molecular karyotpying would provide acontribution to the art.

DISCLOSURE OF THE INVENTION

The present invention discloses that chromosomal analysis by molecularkaryotyping, (for example for the detection of trisomy) can be performedby use of genome wide biallelic marker analysis (e.g. biallelic singlenucleotide polymorphisms (SNPs)) which are distributed throughout thegenome, and which can be readily detected using existing technologies.

This finding is unexpected for several reasons, principally because apriori it would be assumed that a biallelic marker (which provides onlybinary information at a given position on the chromosome) could notpositively identify the presence of three or more different chromosomes.

Nevertheless, as described below, the invention provides that highdensity analysis of closely adjacent SNPs is capable of positivelyidentifying, inter alia, the presence of two chromosomes derived fromone parent and that based on well established assumptions about thefrequency and spacing of recombination events between parentalchromosomes during meiosis, this will allow accurate detection oftrisomy.

Furthermore, the parental origin of the error is identified in each casewhich is not possible by some karyotyping methods.

The methods of the invention therefore do not depend on quantitation ofchromosome specific sequences, as used in some currently availablemethods but rather compare the haplotypes of the test sample with theknown haplotypes of the parents. When combined with existing methods forwhole genome amplification, the methods of the invention areparticularly useful where only relatively small numbers of sample cellsare available for analysis.

Thus in one aspect the invention provides a method of karyotyping atarget cell to detect chromosomal imbalance therein, the methodcomprising:

(a) interrogating closely adjacent biallelic SNPs across the chromosomeof the target cell(b) comparing the result at (a) with the SNP haplotype of paternal andmaternal chromosomes to assemble a notional haplotype of target cellchromosomes of paternal origin and of maternal origin(c) assessing the notional SNP haplotype of target cell chromosomes ofpaternal origin and of maternal origin to detect aneuploidy of thechromosome in the target cell

As set out below, the method can also be used to assess chromosomalrecombination, where it is desired to do so.

The target cell will be one of a sexually reproducing, diploid, species,with a genome in which biallelic SNPs occur at sufficient density toprovide a notional haplotype. Preferably the cell will be an avian,reptilian, or mammalian cell. More preferably the cell is a human ornon-human mammalian cell. The non-human mammal may, for instance be aprimate.

In one embodiment the cell is human at least 2, 3, 4, 5, 6 or all of thehuman chromosomes selected from the following group are assessed: X, Y,22, 21, 18, 16 and 13. Imbalances in any of these chromosomes may beassociated with viable but abnormal pregnancies.

Preferably a total of at least 10, 15 or 20 chromosomes are assessed. Inone embodiment the entire genome of the target cell (e.g. all 24 humanchromosomes) is assessed.

As discussed below, SNPs can be interrogated using conventionaltechniques. This may be preceded by one or more conventionalamplification steps. Preferably, the frequency of the less frequentallele of the biallelic markers in the present maps is at least 10, 20,or 30% (i.e. a heterozygosity rate of at least 0.18, 0.32 or 0.42).

The result of the SNP interrogation will depend on the nucleotides foundat a polymorphic locus on all the copies of the given chromosome in thetarget cell (i.e. normally 2 copies, but may be 0, 1, or 3 where thereare chromosomal abnormalities, or in the case of the X chromosome, 1 or3 copies in a female or 0 or 2 copies in a male, and in the case of theY chromosome, 2 copies in a male embryo). Unless context demandsotherwise, where “a” or “the” chromosome is referred to herein inrespect of SNP genotyping, this refers to typing all the copies of thatchromosome which are present in the target cell.

The next step, which is the assembly of the notional haplotype, will nowbe discussed in more detail.

Assembling a Notional Haplotype

To detect and characterise the presence of chromosomes of paternal ormaternal origin the next step is to assemble a notional haplotype ofeach chromosome. It is termed ‘notional’ herein since it is inferredrather than determined directly, and in certain embodiments the notionalhaplotype of say, a given chromosome of paternal original, may becharacterised in subsequent steps of the method as arising from twocopies of that chromosome of paternal original (in cases of trisomy, forexample).

This notional haplotype may be assembled using particular sub-sets ofSNPs as follows:

Assuming random SNP alleles for each parental chromosome, there are 16different combinations of the four parental alleles for each SNP (Table1).

Based on the haplotypes (i.e. the sequence of SNP alleles) of eachparental chromosome, eight of these combinations can be predicted toresult in genotypes in the test DNA that positively identify thepresence of one out of the four parental chromosomes at that position(‘informative’) and four others will, dependent on results, eitheridentify a pair of chromosomes one from each parent (‘informative’) oridentify two possible combinations of parental chromosomes, out of thefour possible pair-wise combinations (‘semi-informative’).

Therefore in one embodiment of the invention the notional SNP haplotypeof the target cell chromosomes of paternal origin and of maternal originis assembled using:

(i) informative SNP alleles that positively identify which one of thefour paternal and maternal chromosomes, a chromosome in the target cellhas originated from, or positively identify which paternal chromosomeand which maternal chromosome a pair of chromosomes in the target cellhave originated from, and optionally(ii) semi-informative SNP alleles that positively identify which of twopossible combinations of the four possible pair-wise combinations ofpaternal and maternal chromosomes, a pair of chromosomes in the targetcell has originated from.

Characterising Target Cell Chromosomal Origin

In one embodiment of the invention step (c) is performed as follows:

(c1) assessing the notional SNP haplotype of target cell chromosomes ofpaternal origin and of maternal origin and thereby assigning eachchromosome as absent, non-recombinant, recombinant, or present in 2 ormore copies,(c2) deducing aneuploidy of the chromosome in the target cell whereinstep (c1) indicates an imbalance of chromosomes of paternal origin andof maternal origin,

Once the notional haplotype is compiled, the relevant chromosome may beassigned as non-recombinant or recombinant as follows:

Non-recombinant chromosome: the SNP alleles will be identical to one ofthe parental chromosomes along the whole length of the chromosome.Therefore wherever there is an informative combination of SNP genotypesin the parents for that particular chromosome, the results will bepositive whereas, and equally significantly, at SNPs informative for theother chromosome the results will be negative. Semi-informative SNPswill give results consistent with the presence of that particularparental chromosome (see FIG. 2( a)).

Thus in one embodiment a chromosome in the target cell is identified asnon-recombinant wherein the results of its notional SNP haplotype areconsistent with:

(i) its SNP alleles being identical to the SNP alleles of one of the twopaternal chromosomes or one of the two maternal chromosomes along thelength of the chromosome, and(ii) an absence of the SNP alleles of the alternative of the twopaternal or maternal chromosomes.

Recombinant chromosomes: with recombinant chromosomes, the pattern willbe as for non-recombinant chromosomes except that there may be one ormore alternating segments of both that parents chromosomes resultingfrom single, double or higher order recombination between the originalparental chromosomes during the first meiotic division (see FIG. 2 (a)).

In performing the present invention, consideration is given to the factthat where successive informative or semi-informative SNPs indicate aswitch from identifying one parental chromosome to the other (anapparent crossover event) this could be because of (1) an actualcrossover during meiosis (i.e. normal recombination, as referred toabove), (2) the presence of a second parental chromosome (trisomy, asdiscussed hereinafter), or (3) a SNP genotyping error.

Considering SNP genotyping error, as genome wide SNP genotyping methodsare designed to reduce errors to a minimum, in any given instance acrossover event will be the most likely alternative since there isnormally at least one crossover per chromosome arm. A long succession ofinformative and semi-informative SNP results consistent with thatchromosome and not the other parental chromosome would therefore suggestnormal recombination (see FIG. 2).

Therefore in one embodiment, a chromosome in the target cell isidentified as recombinant wherein the results of its notional SNPhaplotype correspond to SNP alleles of both of the two paternalchromosomes or two maternal chromosomes in one or more alternatingsegments consistent with normal recombination between the twochromosomes.

It is known that normal recombination will depend on (1) average, sexand chromosome specific data for the number of recombinations, and (2)interference between chiasmata preventing multiple recombination eventsover short distances.

Therefore in one embodiment, consistency with normal recombination isassessed based on the statistical likelihood of normal recombinationbetween particular adjacent, informative SNP alleles of the two paternalchromosomes or two maternal chromosomes during the first meioticdivision. Preferably the statistical likelihood is assessed based on isone or more of the following criteria:

(i) the average number of recombination events for the specific paternalor maternal chromosome,(ii) the position of the apparent recombination events on eachchromosome arm relative to each other, the centromere and the telomereie the ends of the chromosome arm involved

Multiple chromosomes: if successive informative and semi-informativeSNPs alternate repeatedly and\or apparently randomly between the twoparental chromosomes, it is highly likely that the test DNA is trisomicrather than a series of double crossover or recombination events (FIG.3; FIG. 4).

This is because the pattern of normal recombination is non-random andspecifically the presence of one crossover physically inhibits anothercrossover nearby, a phenomenon known as crossover interference (Bromanand Weber, 2000).

With two non-recombinant chromosomes from one parent the SNP notionalhaplotype result will alternate all along the chromosome. With othercombinations of non-recombinant and recombinant chromosomes, the twoparental haplotypes will be detectable for a segment of the chromosomewhich shows the repetitive, apparently random, alternation.

In terms of the frequencies of ‘normal’ alternating segments, based on alarge experimental data set, Broman and Weber (2000) propose that theprobability of a double crossover between two non-recombinantinformative polymorphisms can be estimated according to the formula:

p=(0.0114d−0.0154)⁴

where p is the probability of a double crossover in an interval dmeasured as genetic distance in centiMorgans (cM) betweennon-recombinant loci.

The probability that one SNP, indicating the presence of the otherparental chromosome (or >1 informative SNP with no interveningcontradictory informative SNPs) is the result of a double crossover istherefore defined by the probability between adjacent flanking SNPsinformative for that chromosome (FIG. 2).

Thus in one embodiment the statistical likelihood of a double crossoverbetween two SNP alleles is calculated according to the formula:

p=(0.0114d−0.0154)⁴

where p is the probability of a double crossover in an interval dmeasured as genetic distance in centiMorgans (cM) between the SNPalleles.

As an example of the operation of this formula, for an average spacingof 0.32 cM (as with the Affymetrix GeneChip 10K system) and n SNPs:

N d (cM) p 10 3.2  1.6 10⁻⁷ 50 16 8.35 10⁻⁴ 100 32 0.015 200 64 0.254

Thus in most cases, the probability of a pattern alternating between thehaplotypes of both chromosomes from one parent at successive informativeand semi-informative SNPs will be very low particularly where thedensity of SNPs analysed is high and generally much lower than thepossibility of genotyping error.

The probability of trisomy is further increased with the number andextent of this alternating pattern which will depend on the number andposition of true crossover events on both of the chromosomes.

In addition to the number of apparent crossovers in the notionalhaplotypes, several other assumptions about the number and distributionof crossovers across the genome (Lynn et al, 2004) may be used inassigning the chromosome as likely recombinant or not:

-   -   Direct counts of the number of chiasmata indicate that the        average total number in males is 50.6 (Hulten, 1974) and in        females 70 (Hulten and Tease, 2003; Tease and Hulten, 2004).    -   Average number of crossovers on individual chromosomes.    -   Distribution of crossovers on individual chromosomes.

Density and Nature of SNPs

Across the whole genome when analysing, for example, 10K SNPs, despitethe high accuracy rate, one or more random genotyping errors causingisolated individual positive results at informative SNPs for the secondparental chromosome may occur.

Therefore in preferred embodiments a threshold number of positive andnegative informative and semi-informative SNPs is set.

In one embodiment at least 5000 and/or 2500 informative and/orsemi-informative SNP alleles, respectively, distributed across the wholegenome are used to assemble the notional SNP haplotype of target cellchromosomes of paternal origin and of maternal origin. However forindividual chromosomes a less number may be sufficient—this can beassessed by those skilled in the art according to the preferred methodof typing and the accuracy associated with it and with any optionalmethod of amplification employed.

In one embodiment the average distance between the interrogated SNPs isless than 0.1, 0.2, 0.3, 0.4 or 0.5 kb.

In one embodiment the average distance between the interrogated SNPs isless than 0.1, 0.2, 0.3, 0.4 or 0.5 cM

Because of allele dropout (ADO), ie the random failure to amplify one ofthe parental alleles, when amplifying the DNA from single or smallnumbers of cells for application in preimplantation genetic diagnosis,SNP genotype analysis may preferably be based in whole or in part onthose results giving a heterozygous result at an informative orsemi-informative SNP. Thus in one embodiment at least 2500 heterozygousinformative SNP alleles (“AB” in Table 1) are used to assemble thenotional SNP haplotype of target cell chromosomes of paternal origin andof maternal origin.

Karyotyping

As noted above, aneuploidy of the chromosome in the target cell isdetected wherein the notional haplotype indicates an imbalance ofchromosomes of paternal origin and of is maternal origin. Details of thedetection strategies of different aneuploidies are as follows:

In one embodiment where the notional SNP haplotype of target cellchromosomes indicates the presence of one chromosome of paternal originand one chromosome of maternal origin and the cell is deduced to benormal diploid in respect of the relevant chromosome.

Nullsomy: In one embodiment, where the notional SNP haplotype of targetcell chromosomes indicates an absence of any chromosome or chromosomesegment of paternal origin and maternal origin, the cell is deduced tobe nullsomic for the relevant chromosome or chromosome segment.

Monosomy: Here, there will be only one chromosome from one parent, butit can be either non-recombinant or recombinant. Monosomy will thereforebe detected in two ways (1) apparent homozygosity (either ‘AA’ or ‘BB’and not ‘AB’) for all SNPs along the chromosome, and (2) identity to thehaplotype for one of the parental chromosomes (non-recombinant) or analternating pattern between the two haplotypes from one parent(consistent with normal recombination between the two chromosomes). Thusin one embodiment, where the notional SNP haplotype of the target cellchromosomes indicates an absence of a chromosome or chromosome segmentof either paternal origin or maternal origin but not both, the cell isdeduced to be monosomic for the relevant chromosome or chromosomesegment.

Monosomies may be detected whether they arise before or afterfertilisation.

Trisomy: As discussed above, where the notional SNP haplotype of targetcell chromosomes indicates the presence of both of the two paternalchromosomes or two maternal chromosomes in a pattern and\or frequencyinconsistent with normal recombination between the two chromosomes, thecell is deduced to be trisomic for all or part of the relevantchromosome or chromosome segment.

Specifically the method is adapted to detect trisomy where pairedchromosomes in each of the paternal or maternal cells differ (which iscommonly the case), and where the two chromosomes of paternal ormaternal origin in the target cell differ over all or part of thechromosome (which would apply to the majority of trisomies i.e. most ofthose arising during meiosis—see FIG. 1( b))) and informative andsemi-informative SNPs appear and are interrogated in the regions whichdiffer (which would typically apply where sufficient density of SNPs areassessed).

It therefore provides a useful tool in detecting aneuploidy in thesecommon situations. Having described certain embodiments of the inventionabove, some particular modes of operation will now be discussed.

Combined and Multiple Detection Strategies

If desired, the invention may be combined with one or more otherkaryotyping strategies.

For example a further step may include quantitation of alleles toincrease the accuracy and resolution of trisomy detection i.e. in oneembodiment the method further comprises confirming the deduction byquantifying the SNPs across the chromosome of the target cell (Meng etal., 2005).

In one embodiment the method further comprises diagnosing the presenceof an inherited genetic disease in the target cell by comparing thenotional SNP haplotype of the target cell with the SNP alleles of thepaternal chromosomes and the maternal chromosomes and one or moreaffected siblings to diagnose the disease in the target cell by linkage.Linkage is a method in which instead of detecting a disease-causing genemutation itself, one or more informative markers such as STRs or SNPs,either close to or within the affected gene, are used to track theaffected copy of the gene by comparison with the markers inherited by anaffected child (Abou-Sleiman et al, 2002). With genome wide SNP analysisof the target cell genotype as discussed above, multiple closely linkedSNPs flanking the affected gene may be analysed permitting highlyaccurate linkage analysis.

In one embodiment the method further comprises diagnosing the presenceof a susceptibility to a common disease or cancer in the target cell bycomparing the notional SNP haplotype with a haplotype known to beassociated with said disease. Such associations are being increasinglyestablished, for example via the “International HapMap Consortium” whichis mapping genome wide variation in SNP haplotypes in the humanpopulation, is to facilitate disease association studies (InternationalHapMap Consortium, 2005). The associations do not per se form part ofthe invention, but the combination of this haplotype analysis with thekaryotyping method described herein forms one aspect of the invention.

Paternal and Maternal Cells and Chromosomal Haplotypes

In one embodiment paternal and maternal cells are provided from blood orbuccal cavity

Analysis of SNPs from related individuals across at least one generationallows the identification of a haplotype for each chromosome inpositions where the alleles are different. Specifically, the haplotypeof SNP alleles can be ascertained by analysing the DNA of each parentand comparing the results with a haploid gamete, child or parent or acombination of these. Those skilled in the art are aware of numerousalgorithms and related software programmes that allow the haplotypes tobe inferred from analysis of diploid individuals e.g. PHASE (Stephensand Donnelly, 2003) and SIMHAP (www.genepi.com.au/simhap).

In one embodiment SNP haplotype of paternal and maternal chromosomes isderived from analysis of the SNP haplotype of cells removed from siblingfertilized embryos following in vitro fertilisation (IVF) followingwhole genome amplification.

In one embodiment SNP haplotype of paternal and maternal chromosomes isderived from analysis of multiple single parental haploid gametesfollowing whole genome amplification.

Where two chromosomes or chromosome segments from one parent are shownto be identical, the method will not be applicable for that chromosomeor segment and alternative methods should be used.

Target Cells

In one embodiment the target cell has been provided from a mammalianembryo which has resulted from IVF. In one embodiment the embryo is apre-implantation embryo (see e.g. Handyside et al, 2004).

Where the invention is applied to animals such as livestock, the embryomay be recovered from the uterus.

In one embodiment the target cell(s) have been provided from a fetus

In one embodiment a number equal to, or at least, 1, 2, 3, 4, or 5 cellsare provided

In one embodiment SNP interrogation is preceded by whole genomeamplification

In one embodiment the whole genome amplification employs isothermalMultiple Displacement Amplification (MDA) which permits whole genomeamplification using the bacteriophage phi29 polymerase for amplificationfrom small numbers of cells (see e.g. Handyside et al, 2004).

Interrogation of SNPs

Preferred markers are biallelic SNPs, which occur throughout the genome(˜10 million per genome, wherein the SNP is defined as >1% variationbetween individuals in a population). Various methods for large scalesingle nucleotide polymorphism (SNP) analysis exist (see Syvanen, 2005,especially Table 1). These include SNPstream (Bell, P. A. et al.SNPstream UHT: ultra-high throughput SNP genotyping for pharmacogenomicsand drug discovery. Biotechniques Suppl., 70-72, 74, 76-77 (2002));Genorama, APEX (Kurg, A. et al. Arrayed primer extension: solid-phasefour-colour DNA resequencing and mutation detection technology. Genet.Test. 4, 1-7 (2000)); GeneChip 100K (Matsuzaki, H. et al. Genotypingover 100,000 SNPs on a pair of oligonucleotide arrays. Nat. Methods 1,109-111 (2004)); Perlegen wafers (Hinds, D. A. et al. Whole-genomepatterns of common DNA variation in three human populations. Science307, 1072-1079 (2005)); Molecular Inversion Probes (Hardenbol, P. et al.Highly multiplexed molecular inversion probe genotyping: Over 10,000targeted SNPs genotyped in a single tube assay. Genome Res. 15, 269-275(2005)); GoldenGate Assay (Fan, J. B. et al. Highly parallel SNPgenotyping. Cold Spring Harb. Symp. On Quant. Biol. LXVII, 69-78(2003)). Other methods include the Illumina “BeadArray”.

A preferred embodiment employs the Affymetrix GeneChip™ 10K Microarrayis designed to analyse 10,000 distributed at an average distance of 0.2Kb across each of 22 chromosomes (see Matsuzaki, H. et al. Parallelgenotyping of over 10,000 SNPs using a one-primer assay on ahigh-density oligonucleotide array. Genome Res. 14, 414-425 (2004))

In the case of oligonucleotide chips, the oligonucleotides that can bebonded to a chip according to the invention will be capable ofdistinguishing biallelic SNPs across the genome. Preferred are 25nucleotide-long oligonucleotides.

Thus in one embodiment the SNPs are interrogated on a “gene” or“oligonucleotide” chip or microarray. As is well known in the art theseare miniaturized vehicles, in most cases made of glass or silicon, onwhose surface oligonucleotides of known sequence are immobilized in anordered grid of high density.

Another preferred embodiment employs the Illumina's “infinium”™ Human-1BeadChip. This system may enable whole-genome genotyping of over 100,000SNP markers, 70% of which are located in exons or within 10 kb oftranscripts (see e.g. Pharmacogenomics (2005) 6(7), 777-782). The systemis based on the random assembly of derivatized microscopic beadsapproximately 3 μm in size) into wells of a patterned substrate, and maypermit specified combinations of SNPs to be interrogated.

Systems

Preferably a system for use in the present invention would comprisesmeans for SNP interrogation plus a programmed storage device or mediumfor causing a computer to analyse the resulting data. The SNPinterrogation data could be stored for later analysis or analysed ‘onthe fly’—as used herein the term “database” covers both types of datasource.

Preferred means for SNP interrogation would be an oligonucloeotide chipwhich would interrogate at least the preferred chromosomes at theappropriate density discussed above. Preferably it would include thewhole genome.

Thus preferred means for SNP interrogation would include:

(i) A high density of biallelic SNPs on chromosomes frequentlyassociated with miscarriage or viable abnormal pregnancies (X, Y, 22,21, 18, 16, 13). The means may interrogate a full polymorphic set onthese chromosomes.(ii) SNPs which are highly heterozygous in the general populationincreasing their informativeness,(iii) Relatively increased density in all the known microdeletionsyndrome regions,(iv) Relatively increased density in regions associated with commonsingle gene defects,(v) Known SNP ‘haplotags’ associated with predisposition to commoncomplex diseases.

The present invention may be implemented with a computer. Typically thiswould include a central processing unit (CPU) connected by a system busor other connecting means to a communication interface, system memory(RAM), non-volatile memory (ROM), and one or more other storage devicessuch as a hard disk drive, a diskette drive, and a CD ROM drive.

The computer also includes a display device, such as a printer, CRTmonitor or an LCD display, and an input device, such as a keyboard,mouse, pen, touch-screen, or voice activation system. The input devicemay receive data directly from the means for SNP interrogation via aninterface (as for example with the Affymetrix system).

The computer stores and executes various programs such as an operatingsystem and application programs.

The computer-usable medium would cause the computer to analysehaplotypes and perform molecular karyotyping to assign parental originalong the length of each chromosome, and report on aneuploidy where thiswas detected. The medium may for example be selected from the groupconsisting of a hard disk a floppy disk, Random Access Memory, Read OnlyMemory and Electrically Eraseable Programable Read Only Memory.

Thus the invention provides a computer-usable medium havingcomputer-readable program code or instructions stored thereon (i.e. aprogrammed storage device) for causing a computer to execute a method todetermine aneuploidy or chromosomal recombination in a target cell, themethod being any one of those discussed herein.

Preferably the method comprises:

(a) accessing a database comprising genotype data obtained from aplurality of closely adjacent biallelic SNP loci present in a chromosomeof the target cell,(b) accessing a database comprising SNP haplotype data of thecorresponding paternal is and maternal chromosomes (i.e. ‘P1’, ‘P2’,‘M1; ‘M2’),(c) comparing target cell SNP data from the database of step (a) withSNP haplotype data from the database of step (b) to assemble a notionalhaplotype of regions of the target cell chromosomes of paternal originand of maternal origin,(d) assessing the notional SNP haplotype of target cell chromosomes ofpaternal origin and of maternal origin to detect aneuploidy orchromosomal recombination of the chromosome in the target cell.

Optionally, each SNP locus of the ‘x’ SNPs of the database in step (b)is assigned a value ‘n’ in accordance with which of the 16 combinationsof four parental SNP alleles is present at that locus, and wherein step(c) comprises assembling a notional haplotype at that locus by comparing

(i) the genotype data for the biallelic SNP at that locus from thedatabase of step (a) and,(ii) the value ‘n’ at that locus from the database of step (b) with,(iii) a chromosomal origin table, and thereby assigning the SNP locus ofthe target cell chromosomes as originating from a paternal or maternalchromosome.

By ‘chromosomal origin table’ is meant a reference set of data by whichthe chromosomal origin (e.g. ‘P1’, ‘P2’, ‘M1; or ‘M2’) can be assignedat that locus based on the values at (i) and (ii). This may correspondto that given in Table 1, last column.

Preferably the notional SNP haplotype of regions of the target cellchromosomes of paternal origin and of maternal origin is assembled usinga subset of the ‘x’ SNP loci from the database in step (b), which subsetconsists of:

(i) informative SNP alleles that positively identify which one of thefour paternal and maternal chromosomes, a chromosome in the target cellhas originated from, or positively identify which paternal chromosomeand which maternal chromosome a pair of chromosomes in the target cellhave originated from, and optionally(ii) semi-informative SNP alleles that positively identify which of twopossible combinations of the four possible pair-wise combinations ofpaternal and maternal chromosomes, a pair of chromosomes in the targetcell has originated from.

Optionally the subset may consist of heterozygous informative SNPalleles.

Optionally the method may comprises storing the notional haplotyperesult obtained for each SNP locus of the ‘x’ SNPs, or a subset thereof.

An example of software (Excel Visual Basic for Applications (VBA) codeI) is listed in Appendix 1, and this identifies the differentcombinations of parental alleles at each SNP and assigns parental originat informative SNP loci. FIG. 4 shows resulting notional haplotypes andthe actual target cell haplotypes as determined independently.

The program may identify the chromosome in the target cell asnon-recombinant wherein the results of its notional SNP haplotype areconsistent with:

(i) its SNP alleles being identical to the SNP alleles of one of the twopaternal chromosomes or one of the two maternal chromosomes along thelength of the chromosome, and(ii) an absence of the SNP alleles of the alternative of the twopaternal or maternal chromosomes.

The program may identify the chromosome in the target cell asrecombinant wherein the results of its notional SNP haplotype correspondto SNP alleles of both of the two paternal chromosomes or two maternalchromosomes in one or more alternating segments consistent with normalrecombination between the two chromosomes.

The program may identify the chromosome in the target cell as trisomicfor the chromosome where the notional SNP haplotype of the target cellchromosome indicates the presence of both of the two paternalchromosomes or two maternal chromosomes in a pattern and\or frequencyinconsistent with normal recombination between the two chromosomes

The program may statistically analyze the likelihood of normalrecombination between the SNP loci based on one or more of the followingcriteria:

(i) a database the average number of recombination events for thespecific paternal or maternal chromosome,(ii) the position of the apparent recombination events on eachchromosome arm relative to each other, the centromere and the telomere

Optionally the program may calculate a numerical measure of probabilityof, for example, trisomy based on this frequency and pattern data.

The program may identify the chromosome in the target cell as nullsomicfor the chromosome where the notional SNP haplotype of target cellchromosome indicates an absence of the chromosome or a segment thereofof both paternal origin and maternal origin.

The program may identify the chromosome in the target cell as monosomicfor the chromosome where the notional SNP haplotype of the target cellchromosome indicates an absence of the chromosome or a segment thereofof paternal origin and maternal origin but not both.

Optionally a threshold number of positive and negative informative andoptionally semi-informative SNPs is set, and a karyotype is determinedonly when this number is exceeded.

The invention also provides a computer programmed to execute a method asdescribed above.

The invention will now be further described with reference to thefollowing non-limiting Figures and Examples. Other embodiments of theinvention will occur to those skilled in the art in the light of these.

The disclosure of all references cited herein, inasmuch as it may beused by those skilled in the art to carry out the invention, is herebyspecifically incorporated herein by cross-reference.

FIGURES

FIG. 1( a): schematic showing normal meiosis I and II

FIG. 1( b): schematic showing non-disjunction during meiosis leading toaneuploidy

FIG. 2: schematic representation of SNP genotype analysis for achromosome pair in test DNA from a fetus or embryo. Each row representsa SNP locus and each column, the haplotypes for the two paternalchromosomes (P1 and P2) and two maternal chromosomes (M1 and M2). Apositive result at an informative SNP is shaded. A negative result at aninformative SNP is marked with ?PX (?MX omitted for clarity). In FIG. 2(a) the segment marked A of the paternal copy of the chromosome isidentified as a double recombinant because multiple consecutiveinformative SNPs positively identify the haplotype of P1 and othersinformative for P2 are negative. The length of the chromosomal segmentand number of crossovers both for this chromosome and overall would alsobe taken into account and because of crossover interference wouldnormally extend over a greater number of SNPs than in this diagrammaticrepresentation. In FIG. 2( b) The maternal chromosome is non-recombinantin this region and only SNPs informative for M1 are positive.

FIG. 3: Schematic representation of SNP genotype analysis for achromosome pair in test DNA from a fetus or embryo as described in FIG.2. In this example, the segment marked B is assigned as trisomic becauseof the positive result for multiple alternating SNPs informative forboth chromosomes. The probability of each individual positive result forP1 being a double recombinant between flanking positive results for P2(marked A) is very small given the average genetic distance between SNPswhen using high density SNP analysis.

FIG. 4: Parental and test offspring genotype data analysed using theExcel VBA code in Appendix 1 illustrating how informative combinationsof biallelic SNPs allow identification the parental origin along achromosome.

FIG. 5: Flowchart illustrating the present invention with multipledetection strategies.

EXAMPLES Example 1 Theoretical Background

During meiosis and the formation of gametes, homologous chromosomes pairand recombine resulting in four chromosomes, which on average willconsist of two non-recombinant and two recombinant chromosomes. Each ofthe resulting chromosomes therefore now has a unique SNP haplotype.

Following fertilisation, each embryo has a unique combination ofhaplotypes from the non-recombinant or recombinant chromosomessegregated to the two gametes from the two parents. In euploid embryos,with the normal pairs of each chromosome, each chromosome will have adistinct haplotype and the parental origin of each chromosome will beidentifiable from the non-recombinant or unique recombinant haplotype.Similarly, trisomy and monosomy will also be detectable.

Table 1 below shows how SNPs can be classified as informative,semi-informative, or non-informative.

TABLE 1 The 16 combinations of parental SNP haplotypes based on a randomdistribution of alleles. Informative combinations identify a parentalhaplotype irrespective of the result. Informative/semi-informativecombinations of alleles identify one of both parents chromosomes or twopossible combinations depending on the genotype of the DNA being tested.Test Informativity # P1 P2 M1 M2 genotype Non- 1 A A A A PA informative2 B B B B BB 3 A A B B AB 4 B B A A AB Informative 5 A B B B AB = P1(all results BB = P2 identify 6 B A B B AB = P2 presence of 1 BB = P1parental 7 B B A B AB = M1 chromosome) BB = M2 8 B B B A AB = M2 BB = M19 B A A A AB = P1 AA = P2 10 A B A A AB = P2 AA = P1 11 A A B A AB = M1AA = M2 12 A A A B AB = M2 AA = M1 Informative/ 13 A B A B AA = P1M1semi- BB = P2M2 informative AB = P1M2 (⅔ possible or P2M1 resultsidentify 14 B A B A AA = P2M2 a pair of BB = P1M1 parental AB = P2M1chromosomes or P1M2 and ⅓ 15 A B B A AA = P1M2 results could be BB =P2M1 either of two AB = P1M1 combinations) or P2M2 16 B A A B AA = P2M1BB = P1M2 AB = P2M2 or P1M1

Example 2 Informativity of SNPs and ADO

In some embodiments when DNA is amplified from single cells, forexample, for preimplantation genetic diagnosis (PGD), one of theparental alleles may fail to amplify at random resulting in alleledropout (ADO). Table 2 below demonstrates that where ADO occurs atinformative SNPs, half of these will be detected because the apparenttest genotype is not possible and therefore the true heterozygous result(“AB”) can be inferred.

TABLE 2 Effect of allele dropout (ADO) at informative SNPs MaternalPaternal (M) chr (P) chr Test genotype and chromosome 1 2 1 2 identifiedB A A A AA = M2 AB = M1 M1 (BB = AB*) A B A A AA = M1 AB = M2 (BB = AB*)A A B A AA = P2 AB = P1 (BB = AB*) A A A B AA = P1 AB = P2 (BB = AB*) AB B B BB = M2 AB = M1 (AA = AB*) B A B B BB = M1 AB = M2 (AA = AB*) B BA B BB = P2 AB = P1 (AA = AB*) B B B A BB = P1 AB = P2 (AA = AB*) *Forthis combination of parental SNPs, the test genotype cannot have twocopies of this allele indicating allele dropout (ADO) i.e. failure toamplify one of the parental alleles at random. The test genotype cantherefore be assumed to be AB. This approach increases the power of thetest when the invention is used in single cell applications such aspreimplantation genetic screening.

Example 3 Combined SNP Quantitative and Sequence Based Analysis

If relative quantitation of each SNP allele is achieved, the normaldisomic genotype combinations, “AA”, “AB” and “BB”, are supplemented by“A” and “B” in monosomy and “AAA”, “BBB”, “AAB” and “ABB”.

Table 3 demonstrates the extra information that is available bycombining genotyping and quantitation of SNPs. While these possiblecombination of SNP alleles genotyping may be uninformative, quantitationwould identify the chromosome as trisomic even though the parentalorigin is unknown in the first two examples.

TABLE 3 Combined genotyping and quantitation of SNPs Maternal (M) chrPaternal (P) chr Test genotype and chromosome 1 2 1 2 identified A A A AAAA = Trisomic M1 M2 or P1 P2 B B B B BBB = Trisomic M1 M2 or P1 P2 A AB B AAB = M1 M2 P1 or P2 ABB = M1 or M2 P1 P2 B B A A AAB = M1 or M2 P1P2 BBA = M1 M2 P1 or P2 A B A B AAB = M1 P1 + M2 or P2 ABB = M2 P2 + M1or P1 B A A B AAB = M2 P1 + M1 or P2 ABB = M1 P2 + M2 or P1 A B B A AAB= M1 P2 + M2 or P1 BBA = M2 P1 + M1 or P2 B A B A AAB = M2 P2 + M1 or P1BBA = M1 P1 + M2 or P2

Example 4 Use of Multiple Displacement Amplification (MDA)

A Microsoft Excel VBA macro (SNP analysis (1)) to analyse thesecombinations of SNPs and test genotypes is set out below in Appendix 1.

The results are shown in FIG. 4. The data is test genotype analysis ofSNPs across chromosome 21. Maternal and paternal haplotypes have beencreated based on an individual genotyped using the Affymetrix GeneChip10K microarray. At some SNPs the alleles could not be reliablyidentified (“No call”). Otherwise, for illustration purposes, the testgenotype is assumed to be 100% accurate and is based on the actual testhaplotypes shown. The numbered parental haplotype combinations are asdefined in Table 1. In this example only fully informative SNPscombinations (5-12) have been used to identify the test haplotypes. Inaddition, semi-informative combinations would be used and theinformation further analysed to distinguish double recombination fromtrisomy or genotyping errors. Two examples are shown—one normal disomicand one trisomic for chromosome 21. The black circles indicate thecritical SNP results that indicate this test sample is trisomic i.e.they demonstrate the presence of alternating segments which are notconsistent with normal recombination between the two chromosomes.

Example 5 Prenatal Diagnosis Following CVS or Amniocentesis

Current methods of analysis for chromosomes include karyotyping,fluorescent in situ is hybridisation (FISH) for a restricted number ofchromosome pairs or quantitative fluorescent PCR. SNP profilingaccording to the present invention combines detection of aneuploidy,deletions and unbalanced translocations.

Current methods for detection of single gene defects combine mutationdetection (requiring identification of the mutation) and informativelinked markers in some cases requiring prior linkage analysis and testdevelopment. Because of the density of SNPs analysable linkage tocombinations of SNPs will also be possible in many cases.

Example 6 Preimplantation Genetic Diagnosis Following IVF

Preimplantation genetic diagnosis (PGD) requires analysis of single orsmall numbers of cells removed from each early embryo and, if possible,within 36-72 h, so that embryos identified as unaffected can betransferred without cryopreservation.

Current methods for aneuploidy include sequential FISH for analysis of 9chromosomes, comparative genomic hybridisation of all chromosomesrequiring cryopreservation and multiplex fluorescent PCR. Eachreciprocal translocation and each type of Robertsonian translocationrequire the development of a specific strategy. Current methods forsingle gene detection include mutation detection and linkage analysis bymultiplex fluorescent PCR.

TABLE 4 Advantages of the present invention in prenatal diagnosis andpreimplantation genetic diagnosis. Advantages Prenatal diagnosisUniversal screen for aneuploidy (possible exceptions) High resolutiondetection of unbalanced transloca- tions and common deletionsPreimplantation Universal screen for aneuploidy (including trans-genetic diagnosis locations) combined with single gene defect linkagedetection No requirement for test development Identifies parental originof aneuploidy

REFERENCES

-   Handyside et al (2004) Isothermal whole genome amplification from    single and small numbers of cells: a new era for preimplantation    genetic diagnosis of inherited disease. Mol Hum Reprod 10, 767-772.-   Syvanen, A C (2005) Toward genome wide SNP genotyping. Nature    Genetics 37, S5-S10.-   Broman, K. W. and Weber, J. L. (2000) Characterisation of human    crossover interference. Am J Hum Genet 66, 1911-1926.-   Hulten, maternal (1974) Chiasma distribution at diakinesis in the    normal human male. Hereditas 76, 55-78.-   Hulten M A and Tease C (2003) Genetic maps: direct meiotic analysis.    In: Cooper D N (ed) Encyclopaedia of the Human Genome Nature    Publishing Group, London-   Lynn, A, Ashley, T and Hassold, T (2004) Variation in human meiotic    recombination. Annu Rev Genomics Hum Genet 5, 317-349.-   Tease C and Hulten M A (2004) Inter-sex variation in synaptonemal    complex lengths largely determine the different recombination rates    in male and female germ cells. Cytogenet Genome Res 107, 208-215.-   Meng H, Hager K and Gruen J R (2005) Detection of Turner syndrome    using high-throughput quantitative genotyping. J Clin Endocrinol    Metab 90, 3419-3422.-   International HapMap Consortium (2005) A haplotype map of the human    genome. Nature 437, 1299-1320.-   Abou-Sleiman P M, Apessos A, Harper J C, Serhal P, Winston R M and    Delhanty J D (2002) First application of preimplantation genetic    diagnosis to neurofibromatosis type 2 (NF2). Prenatal Diagnosis 22,    519-524.-   Stephens M and Donnelly P (2003) A comparison of Bayesian methods    for haplotype reconstruction from population genotype data. Am J Hum    Genet 73, 1162-1169.

APPENDIX 1 Microsoft Excel VBA macro: SNP analysis (1) Private SubCommandButton1_Click( ) For i = 1 To X (no. of SNPs analysed) IfCells(i, 1) = “A” And Cells(i, 2) = “A” And Cells(i, 3) = “A” AndCells(i, 4) = “A” Then Cells(i, 5).Value = 1 If Cells(i, 1) = “B” AndCells(i, 2) = “B” And Cells(i, 3) = “B” And Cells(i, 4) = “B” ThenCells(i, 5).Value = 2 If Cells(i, 1) = “A” And Cells(i, 2) = “A” AndCells(i, 3) = “B” And Cells(i, 4) = “B” Then Cells(i, 5).Value = 3 IfCells(i, 1) = “B” And Cells(i, 2) = “B” And Cells(i, 3) = “A” AndCells(i, 4) = “A” Then Cells(i, 5).Value = 4 If Cells(i, 1) = “A” AndCells(i, 2) = “B” And Cells(i, 3) = “A” And Cells(i, 4) = “A” ThenCells(i, 5).Value = 5 If Cells(i, 1) = “A” And Cells(i, 2) = “B” AndCells(i, 3) = “B” And Cells(i, 4) = “B” Then Cells(i, 5).Value = 6 IfCells(i, 1) = “B” And Cells(i, 2) = “A” And Cells(i, 3) = “A” AndCells(i, 4) = “A” Then Cells(i, 5).Value = 7 If Cells(i, 1) = “B” AndCells(i, 2) = “A” And Cells(i, 3) = “B” And Cells(i, 4) = “B” ThenCells(i, 5).Value = 8 If Cells(i, 1) = “A” And Cells(i, 2) = “A” AndCells(i, 3) = “A” And Cells(i, 4) = “B” Then Cells(i, 5).Value = 9 IfCells(i, 1) = “B” And Cells(i, 2) = “B” And Cells(i, 3) = “A” AndCells(i, 4) = “B” Then Cells(i, 5).Value = 10 If Cells(i, 1) = “A” AndCells(i, 2) = “A” And Cells(i, 3) = “B” And Cells(i, 4) = “A” ThenCells(i, 5).Value = 11 If Cells(i, 1) = “B” And Cells(i, 2) = “B” AndCells(i, 3) = “B” And Cells(i, 4) = “A” Then Cells(i, 5).Value = 12 IfCells(i, 1) = “A” And Cells(i, 2) = “B” And Cells(i, 3) = “A” AndCells(i, 4) = “B” Then Cells(i, 5).Value = 13 If Cells(i, 1) = “B” AndCells(i, 2) = “A” And Cells(i, 3) = “A” And Cells(i, 4) = “B” ThenCells(i, 5).Value = 14 If Cells(i, 1) = “A” And Cells(i, 2) = “B” AndCells(i, 3) = “B” And Cells(i, 4) = “A” Then Cells(i, 5).Value = 15 IfCells(i, 1) = “B” And Cells(i, 2) = “A” And Cells(i, 3) = “B” AndCells(i, 4) = “A” Then Cells(i, 5).Value = 16 Next i For i = 1 To X IfCells(i, 5) = 5 And Cells(i, 6) = “AB” Then Cells(i, 9).Interior.Color =RGB(200, 0, 0) If Cells(i, 5) = 5 And Cells(i, 6) = “BB” Then Cells(i,9).Interior.Color = RGB(200, 0, 0) If Cells(i, 5) = 5 And Cells(i, 6) =“AA” Then Cells(i, 8).Interior.Color = RGB(200, 0, 0) If Cells(i, 5) = 6And Cells(i, 6) = “AB” Then Cells(i, 8).Interior.Color = RGB(200, 0, 0)If Cells(i, 5) = 6 And Cells(i, 6) = “AA” Then Cells(i,8).Interior.Color = RGB(200, 0, 0) If Cells(i, 5) = 6 And Cells(i, 6) =“BB” Then Cells(i, 9).Interior.Color = RGB(200, 0, 0) If Cells(i, 5) = 7And Cells(i, 6) = “AB” Then Cells(i, 8).Interior.Color = RGB(200, 0, 0)If Cells(i, 5) = 7 And Cells(i, 6) = “BB” Then Cells(i,8).Interior.Color = RGB(200, 0, 0) If Cells(i, 5) = 7 And Cells(i, 6) =“AA” Then Cells(i, 9).Interior.Color = RGB(200, 0, 0) If Cells(i, 5) = 8And Cells(i, 6) = “AB” Then Cells(i, 9).Interior.Color = RGB(200, 0, 0)If Cells(i, 5) = 8 And Cells(i, 6) = “AA” Then Cells(i,9).Interior.Color = RGB(200, 0, 0) If Cells(i, 5) = 8 And Cells(i, 6) =“BB” Then Cells(i, 8).Interior.Color = RGB(200, 0, 0) If Cells(i, 5) = 9And Cells(i, 6) = “AB” Then Cells(i, 11).Interior.Color = RGB(0, 0, 200)If Cells(i, 5) = 9 And Cells(i, 6) = “BB” Then Cells(i,11).Interior.Color = RGB(0, 0, 200) If Cells(i, 5) = 9 And Cells(i, 6) =“AA” Then Cells(i, 10).Interior.Color = RGB(0, 0, 200) If Cells(i, 5) =10 And Cells(i, 6) = “AB” Then Cells(i, 10).Interior.Color = RGB(0, 0,200) If Cells(i, 5) = 10 And Cells(i, 6) = “AA” Then Cells(i,10).Interior.Color = RGB(0, 0, 200) If Cells(i, 5) = 10 And Cells(i, 6)= “BB” Then Cells(i, 11).Interior.Color = RGB(0, 0, 200) If Cells(i, 5)= 11 And Cells(i, 6) = “AB” Then Cells(i, 10).Interior.Color = RGB(0, 0,200) If Cells(i, 5) = 11 And Cells(i, 6) = “BB” Then Cells(i,10).Interior.Color = RGB(0, 0, 200) If Cells(i, 5) = 11 And Cells(i, 6)= “AA” Then Cells(i, 11).Interior.Color = RGB(0, 0, 200) If Cells(i, 5)= 12 And Cells(i, 6) = “AB” Then Cells(i, 11).Interior.Color = RGB(0, 0,200) If Cells(i, 5) = 12 And Cells(i, 6) = “AA” Then Cells(i,11).Interior.Color = RGB(0, 0, 200) If Cells(i, 5) = 12 And Cells(i, 6)= “BB” Then Cells(i, 10).Interior.Color = RGB(0, 0, 200) Next i End Sub

1. A method of karyotyping a human target cell to detect chromosomalimbalance therein, the method comprising: (a) interrogating closelyadjacent biallelic SNPs across the chromosome of the target cell (b)comparing the result at (a) with the SNP haplotype of paternal andmaternal chromosomes to assemble a notional haplotype of target cellchromosomes of paternal origin and of maternal origin (c) assessing thenotional SNP haplotype of target cell chromosomes of paternal origin andof maternal origin to detect aneuploidy of the chromosome in the targetcell, wherein the notional SNP haplotype of the target cell chromosomesof paternal origin and of maternal origin is assembled in step (b)using: (i) informative SNP alleles that positively identify from whichone of the four paternal and maternal chromosomes a chromosome in thetarget cell has originated, or positively identify from which paternalchromosome and which maternal chromosome a pair of chromosomes in thetarget cell has originated, and optionally (ii) semi-informative SNPalleles that Positively identify from which of two possible combinationsof the four possible pair-wise combinations of paternal and maternalchromosomes a pair of chromosomes in the target cell has originated. 2.(canceled)
 3. A method as claimed in claim 1 wherein: (i) at least 2, 3,4, 5, 6, or 7 of the chromosomes selected from the group consisting of:X, Y, 22, 21, 18, 16 and 13 are interrogated, or (ii) all 24 chromosomesare interrogated. 4.-6. (canceled)
 7. A method as claimed in claim 1wherein aneuploidy of the chromosome in the target cell is detected instep (c) by: (c1) assessing the notional SNP haplotype of target cellchromosomes of paternal origin and of maternal origin and therebyassigning each chromosome as recombinant or non-recombinant and\orpresent in 0, 1, or more copies, (c2) deducing aneuploidy of thechromosome in the target cell wherein step (c1) indicates an imbalanceof chromosomes of paternal origin and of maternal origin.
 8. A method asclaimed in claim 1 wherein a chromosome in the target cell is identifiedas non-recombinant wherein the results of its notional SNP haplotype areconsistent with: (i) its SNP alleles being identical to the SNP allelesof one of the two paternal chromosomes or one of the two maternalchromosomes along the length of the chromosome, and (ii) an absence ofthe SNP alleles of the alternative of the two paternal or maternalchromosomes, and wherein a chromosome in the target cell is identifiedas recombinant wherein the results of its notional SNP haplotypecorrespond to SNP alleles of both of the two paternal chromosomes or twomaternal chromosomes in one or more alternating segments consistent withnormal recombination between the two chromosomes.
 9. (canceled)
 10. Amethod as claimed in claim 1 wherein the notional SNP haplotype oftarget cell chromosomes indicates the presence of both of the twopaternal chromosomes or two maternal chromosomes in a pattern and\orfrequency inconsistent with normal recombination between the twochromosomes, the cell is deduced to be trisomic for all or part of therelevant chromosome or chromosome segment.
 11. A method as claimed inclaim 10 wherein consistency with normal recombination is assessed basedon the statistical likelihood of normal recombination between particularadjacent, informative SNP alleles of the two paternal chromosomes or twomaternal chromosomes during the first meiotic division, and wherein thestatistical likelihood is assessed based on one or more of the followingcriteria: (i) the average number of recombination events for thespecific paternal or maternal chromosome, (ii) distance between apparentrecombination events on each chromosome arm, and their position relativeto each other, the centromere and the telomere. 12.-13. (canceled)
 14. Amethod as claimed in claim 1 wherein the notional SNP haplotype oftarget cell chromosomes indicates (i) the presence of one chromosome ofpaternal origin and one chromosome of maternal origin and the cell isdeduced to be normal diploid in respect of the relevant chromosome, orindicates (ii) an absence of any chromosome or chromosome segment ofpaternal origin and maternal origin, the cell is deduced to be nullsomicfor the relevant chromosome or chromosome segment, or indicates (iii) anabsence of a chromosome or chromosome segment of either paternal originor maternal origin but not both, the cell is deduced to be monosomic forthe relevant chromosome or chromosome segment. 15.-16. (canceled)
 17. Amethod as claimed in claim 1 wherein the average distance between theinterrogated SNPs is less than 0.1, 0.2, 0.3, 0.4 or 0.5 kb or less than0.1, 0.2, 0.3, 0.4 or 0.5 cM.
 18. (canceled)
 19. A method as claimed inclaim 1 further comprising the step of quantitation of interrogated SNPalleles.
 20. A method as claimed in claim 1 further comprising: (i)diagnosing the presence of an inherited genetic disease in the targetcell by comparing the notional SNP haplotype of the target cell with theSNP alleles of the paternal chromosomes and the maternal chromosomes andone or more affected siblings to diagnose the disease in the target cellby linkage and\or (ii) diagnosing susceptibility to a common disease orcancer in the target cell by comparing the notional SNP haplotype with ahaplotype known to be associated with said disease.
 21. (canceled)
 22. Amethod as claimed in claim 1 wherein the SNP haplotype of paternal andmaternal chromosomes is derived from analysis of the SNP haplotype ofcells removed from sibling fertilized embryos following in vitrofertilisation (IVF) following whole genome amplification or fromanalysis of multiple single parental haploid gametes following wholegenome amplification.
 23. (canceled)
 24. A method as claimed in claim 1wherein the target cell has been provided from a mammalian embryo whichhas optionally resulted from IVF and is optionally a Pre-implantationembryo. 25.-26. (canceled)
 27. A method as claimed in claim 1 wherein anumber equal to 1, 2, 3, 4, or 5 target cells are provided and tested.28. A method as claimed in claim 1 wherein SNP interrogation is precededby whole genome amplification.
 29. (canceled)
 30. A method as claimed inclaim 1 wherein the SNP interrogation is performed by means of anoligonucleotide chip.
 31. A computer-usable medium havingcomputer-readable program code stored thereon for causing a computer toexecute a method to determine aneuploidy or chromosomal recombination ina target cell, which method is the method of claim
 1. 32. Acomputer-usable medium having computer-readable program code storedthereon for causing a computer to execute a method to determineaneuploidy or chromosomal recombination in a target cell, which methodcomprises: (a) accessing a database comprising genotype data obtainedfrom a plurality of closely adjacent biallelic SNP loci present in achromosome of the target cell, (b) accessing a database comprising SNPhaplotype data of the corresponding paternal and maternal chromosomes,(c) comparing target cell SNP data from the database of step (a) withSNP haplotype data from the database of step (b) to assemble a notionalhaplotype of regions of the target cell chromosomes of paternal originand of maternal origin, (d) assessing the notional SNP haplotype oftarget cell chromosomes of paternal origin and of maternal origin todetermine aneuploidy or chromosomal recombination of the chromosome inthe target cell.
 33. A computer-usable medium as claimed in claim 32wherein each SNP locus of the ‘x’ SNPs of the database in step (b) isassigned a value ‘n’ in accordance with which of the 16 combinations offour parental SNP alleles is present at that locus, and wherein step (c)comprises assembling a notional haplotype at that locus by comparing:(i) the genotype data for the biallelic SNP at that locus from thedatabase of step (a) and, (ii) the value ‘n’ at that locus from thedatabase of step (b) with, (iii) a chromosomal origin table, and therebyassigning the locus of the target cell chromosomes as originating from apaternal or maternal chromosome.
 34. A computer-usable medium as claimedin claim 32 wherein the notional SNP haplotype of regions of the targetcell chromosomes of paternal origin and of maternal origin is assembledusing a subset of the ‘x’ SNP loci from the database in step (b), whichsubset consists of: (i) informative SNP alleles that positively identifywhich one of the four paternal and maternal chromosomes, a chromosome inthe target cell has originated from, or positively identify whichpaternal chromosome and which maternal chromosome a pair of chromosomesin the target cell have originated from, and optionally (ii)semi-informative SNP alleles that positively identify which of twopossible combinations of the four possible pair-wise combinations ofpaternal and maternal chromosomes, a pair of chromosomes in the targetcell has originated from, wherein a threshold number of positive andnegative informative and optionally semi-informative SNPs is set, and akaryotype is determined only when this number is exceeded. 35.-37.(canceled)
 38. A computer-usable medium as claimed in claim 32 whereinthe chromosome in the target cell is identified as non-recombinantwherein the results of its notional SNP haplotype are consistent with:(i) its SNP alleles being identical to the SNP alleles of one of the twopaternal chromosomes or one of the two maternal chromosomes along thelength of the chromosome, and (ii) an absence of the SNP alleles of thealternative of the two paternal or maternal chromosomes, and wherein thechromosome in the target cell is identified as recombinant wherein theresults of its notional SNP haplotype correspond to SNP alleles of bothof the two paternal chromosomes or two maternal chromosomes in one ormore alternating segments consistent with normal recombination betweenthe two chromosomes.
 39. (canceled)
 40. A computer-usable medium asclaimed in claim 32 wherein: (i) the chromosome in the target cell isidentified as trisomic for the chromosome where the notional SNPhaplotype of the target cell chromosome indicates the presence of bothof the two paternal chromosomes or two maternal chromosomes in a patternand\or frequency inconsistent with normal recombination between the twochromosomes; (ii) the chromosome in the target cell is identified asnullsomic for the chromosome where the notional SNP haplotype of targetcell chromosome indicates an absence of the chromosome or a segmentthereof of both paternal origin and maternal origin; or (iii) thechromosome in the target cell is identified as monosomic for thechromosome where the notional SNP haplotype of the target cellchromosome indicates an absence of the chromosome or a segment thereofof paternal origin and maternal origin but not both. 41.-45. (canceled)46. A system for karyotyping a target cell to detect chromosomalimbalance therein, the system comprising: (i) means for interrogatingclosely adjacent biallelic SNPs across the chromosome of the targetcell, which is an oligonucleotide chip, and (ii) a computer programmedto execute a method as claimed in claim
 1. 47.-49. (canceled)
 50. Asystem for karyotyping a target cell to detect chromosomal imbalancetherein, the system comprising: (i) means for interrogating closelyadjacent biallelic SNPs across the chromosome of the target cell, whichis an oligonucleotide chip, and (ii) a computer programmed with thecomputer-usable medium as claimed in claim 31.